Reactive oxidants, such as hydrogen peroxide (H202) and peroxynitrite (ONOO-), are strongly associated with lung disease. Yet, the cellular and molecular mechanisms that link oxidant exposure to the development of lung disease are poorly understood. One direction of our studies has been to unravel the molecular mechanisms of oxidant-induced ceramide mediated apoptosis. The other direction, which is the focus of this application, aims to elucidate the molecular interactions between reactive oxidants and ErbB1, the epidermal growth factor receptor (EGFR), that drive aberrant growth control leading to airway epithelial hyperplasia. We propose that reactive oxidants act as independent participants in the "input layer" of signals to the ErbB1 receptor but with a different outcome than that of physiologic ligands such as EGF. We have shown that in contrast to ligand-induced dimerization and phn._ohorylation of EGFR, H202 exposure results in an aberrantly phosphorylated EGFR, and ONOO- exposure generates a covalently cross-linked receptor. Moreover, we have recently observed that H202 inhibits the association of the ubiquitin E3 ligase, c-Cbl, with EGFR, and thus prevents the receptor's ubiquitination. These preliminary studies lead us to hypothesize that oxidant-induced aberrant phosphorylation of EGFR prevents its ubiquitination by c-Cbl and proteasomal (or lysosomal) down-regulation. This could result in continuous pro-growth signaling. To test this, we will determine the structure/function of EGFR following exposure to reactive oxidants. We will first map the specific sites of phosphorylation or dimerization targeted by reactive oxidants. Then, we will construct EGFR mutants of the affected sites and elucidate their roles in EGFR function. We will specifically test the hypothesis that by targeting aberrant phosphorylation sites, reactive oxidants prevent EGFR association with the proteasomal component, c-Cbl, and preclude ubiquitination and down-regulation of the receptor, thereby leading to airway epithelial hyperplasia. Structure/function characterization of oxidant-mediated alterations in EGFR sites of phosphorylation (or dimerization), followed by construction of the cognate tyrosine mutants, are important milestones that would link oxidant-specific changes to lung hyperplasia at a molecular level. In the long run, this approach should indicate precise targets for clinical intervention to control hyperplasia of epithelial cells in pulmonarydiseases.